SUPPLEMENTARY INFORMATION (SI) FIGURES AND TABLES
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1 SUPPLEMENTARY INFORMATION (SI) FIGURES AND TABLES 1 Title: Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling Authors: Ralph Adams 1,#, Rebecca J. Burnley 1,#, Chiara R. Valenzano 1,#, Omar Qureshi 1,CarlDoyle 1, Simon Lumb 1, Maria del Carmen Lopez 1,^, Robert Griffin 1, David McMillan 1, Richard D. Taylor 1,Chris Meier 1, Prashant Mori 1, Laura M. Griffin 1, Ulrich Wernery 2, Jörg Kinne 2, Stephen Rapecki 1, Terry S. Baker 1, Alastair D. G. Lawson 1, Michael Wright 1, and Anna Ettorre 1, * Affiliations: 1 New Medicines, UCB-Celltech, 208 Bath Road, SL1 3WE, Slough UK; 2 Central Veterinary Research Laboratory, P.O.Box 597, Dubai, United Arab Emirates. # These authors contributed equally to the work; * corresponding author.
2 2 SI Figure 1 S22 S22 Y27 Y27 Y32 Y32 SI Figure 1. Differential hydrogen bonding in the two copies of the crystallographic asymmetric unit. There are two copies of the VHH6 IL-6 gp80 6 complex in the crystallographic asymmetric unit. In one copy, IL-6 (green) residue Ser22 forms a hydrogen bond with VHH6 (orange) residue Tyr32 (left), and in the second copy, Ser22 forms a hydrogen bond with VHH6 residue Tyr27 (right).
3 SI Figure 2 IL 6 Site 1 3 gp130 VHH6 Site 2 gp80 SI Figure 2. Model of crystal structure of VHH6 IL-6 gp80 superimposed with gp130. Superimposition of IL-6 (green) and gp80 (blue) from the signaling complex, IL-6 gp80 gp130 (PDB code 1P9M), showed low r.m.s.d. values of 1.4±0.2 Å and 1.35±0.05 Å respectively, indicating that VHH6 (orange) holds IL-6 and gp80 together in a form able to bind gp130 (cyan).
4 4 SI Figure 3 SI Figure 3. Stabilization of the IL-6 gp80 complex by VHH6 promotes higher and sustained STAT3 phosphorylation signal in HUVECs comparable to FusionIL-6 fusion protein. HUVECs were treated with FusionIL-6, IL-6+gp80 and VHH6+IL-6+gp80 as described in Materials and Methods. STAT3 phosphorylation signal was quantified at different time points using the spot total intensity per object parameter as described in Materials and Methods. Statistical analysis of pstat3 signal from three replicates was performed for each time point (statistical significance: *=p 0.05; **=p 0.01 and ***=p 0.001). Unlike IL-6+gp80, in the presence of VHH6 or when FusionIL-6 was added to the culture, pstat3 was increased at earlier (30 min) and later time points (180 min and 360 min). y-axis: pstat3 fluorescence expressed as spot total intensity per object; x-axis: time (min).
5 SI Figure 4 VHH6+ IL 6+ gp80 IL 6+ gp SI Figure 4. Transcriptomic analysis of HUVECs treated with VHH6+IL-6+gp80 confirms selective up-regulation of proinflammatory genes. Three different batches of HUVECs were analyzed at 30, 180 and 360 min. VHH6+IL-6+gp80 (sample) were compared to IL-6+gp80 (control). Data were analyzed using the RT 2 Profiler PCR array web based data analysis template v3.5 ( pcr/arrayanalysis) and changes in gene expression changes were calculated using the C t method with normalization of the raw data to housekeeping genes. A heat map was generated using Genedata s Analyst software. Genes in grey-black are down-regulated, while genes in yellow are up-regulated. Gene regulation for each sample is expressed as fold change compared to control.
6 6 SI Table 1 Table 1A: Data collection and refinement statistics (molecular replacement) Junctional Epitope Antibody in complex with IL-6 and gp80 Data collection Space group C Cell dimensions a, b, c (Å) , 67.80, ( ) 90.00, , Resolution (Å) ( )* R meas (%) CC 1/2 (%) 7.2(50.1) 99.9(91.5) I / I 20.88(3.97) Completeness (%) 99.4(96.9) Redundancy 7.5(7.5) Refinement Resolution (Å) No. reflections 34,870 R work /R free / No. atoms Protein 7590 Water 111 B-factors Protein Water R.m.s. deviations Bond lengths (Å) Bond angles ( ) *Values in parentheses are for highest-resolution shell.
7 7 SI Table 1B: Single mutants of VHH6 kd (1/s) wtvhh6 N74A K113A Y32A S101A Y27A 3.6E E E E E E-04 SI Table 1C: Double mutants of VHH6 kd (1/s) wtvhh6 Y27A Y32A N74A S101A K113A S101A K113S S101A 3.6E E E E E-04 SI Table 1. Additional data supporting the junctional epitope nature of VHH6. (A) Data collection and refinement statistics (molecular replacement). To assess the contributions of the side chains to binding, alanine-scanning was carried out. The dissociation rate of single (B) and double mutants (C) were assessed using SPR.
8 8 SI Table 2: Peptides identified and quantified in IL-6 and gp80 by HDX-MS SI Table 2A: Peptide identified in IL-6 Start Residue End Residue Sequence 2 timepoints with p< APVPPGEDSKDV 3 23 PPGEDSKDVAAPHRQPLTSSE SSERIDKQ * SSERIDKQIRY * SERIDKQIRY * RIDKQIRYIL * * IDKQIRYILDGI * * IRYILDGISAL ALRKETCNKSNM # LRKETCNKSNM # RKETCNKSNM # RKETCNKSNMCE KETCNKSNMCESSKEALAENN # ETCNKSNMCESSKEA SSKEALAENNLNLPKMAEKDGCF Q SKEALAENNLN SKEALAENNLNLPKMAEKDGCFQ EALAENNLNLP AENNLNLPKM AENNLNLPKMAEKDGCF * * ENNLNLPKMAEKDGCFQSGFNE * NNLNLPKM NNLNLPKMAEK NNLNLPKMAEKDGCFQSGFNEET * AEKDGCF * * KDGCFQSGFNEE * QSGFNEETCL VKIITGLL VKIITGLLE IITGLLE LEYLQNRFESS EYLQNRFESSE EYLQNRFESSEE EYLQNRFESSEEQA 4 timepoints with p<0.01
9 EYLQNRFESSEEQARAVQMSTK YLQNRFESSEE YLQNRFESSEEQA RFESSEEQARA ESSEEQA ESSEEQARAVQ EEQARAVQ VQMSTKVL VQMSTKVL KVLIQFL IQFLQKKAKNLDA LQKKAKNL * LQKKAKNLDA * LQKKAKNLDAITTPDPTTN * LQKKAKNLDAITTPDPTTNA LQKKAKNLDAITTPDPTTNASL * KAKNLDAITTPDPTT ITTPDPTTNASL * * LTKLQAQN LTKLQAQN LTKLQAQNQWL LTKLQAQNQWLQD QAQNQWL QDMTTHL * QDMTTHLIL * MTTHLIL * ILRSFKE * * ILRSFKEF * * ILRSFKEFL * RSFKEFL * * RSFKEFLQSSL * * 9
10 10 SI Table 2B: SI Table 2A: Peptide identified in gp80 Start Residue End Sequence Residue HENLYFQGLAPRRCPAQEVAR GLAPRRCPAQ GLAPRRCPAQE * GLAPRRCPAQEVA * RCPAQEVARGAGAGDVPPE AQEVARGAGAGDVPPEEPQLSC * QEVARGAGAGDVPP EVARGAGAGDVP * VARGAGAGDVPPEEPQL * RGAGAGDVPPEEPQL * DVPPEEPQLSCFRKSPLSN * PPEEPQL FRKSPLSNV KSPLSNVVCEWGPRSTPSL VVCEWGPRSTPSL VCEWGPRSTPSL WGPRSTPSLTTKAVL TTKAVLL LVRKFQNSPAED VRKFQNSPAED QNSPAEDFQEP AEDFQEPCQYSQESQKFSCQ DFQEPCQ CQYSQESQKFSCQLAVPEG QYSQESQKFSCQLAVPEG YSQESQKFSCQ SQKFSCQ CQLAVPEGDSSFYI LAVPEGDSSF * PEGDSSF * CVASSVGSKF ASSVGSKF LQPDPPANITV * ANITVTAVAR TAVARNPRWLS * AVARNPRW VARNPRW VARNPRWLSV * 2 timepoints with p< timepoints with p<0.01
11 VARNPRWLSVT * TWQDPHSWNSSF * DPHSWNSSF YRLRFEL ELRYRAERSKT RYRAERSKT SKTFTTWMVKDLQH * * MVKDLQHHAVIHD * MVKDLQHHAVIHDAWSG * * MVKDLQHHAVIHDAWSGLRHVVQL * SGLRHVVQL LRHVVQL RHVVQLRA RAQEEFGQGE * * RAQEEFGQGEW * * RAQEEFGQGEWSE * * EEFGQGEWSEWSPEAMGTP GQGEWSEWSPEAMGTPW WSEWSPEA * WSEWSPEAMGTPWTE WSEWSPEAMGTPWTESRSPP SEWSPEA SEWSPEAMGTPWTE SEWSPEAMGTPWTESRSPP WSPEAMGTPWTE PEAMGTPWTESRSPP MGTPWTE SI Table 2: Additional data supporting HDX-MS. List of peptides identified in IL-6 (A) and gp80 (B) and quantified by HDX-MS. Asterisk (*) indicates deuterium uptake increasing the presence of VHH6; hash (#) indicates deuterium uptake decreasing in the presence of VHH6.
12 12 SI Table 3: SPR analysis IL-6 immobilized kd (1/s) SE (kd) KD (M) SE (KD) gp E E E-09 gp80+vhh6 (2 M) 1.94E E-07 NA gp80 immobilized kd (1/s) SE (kd) KD (M) SE (KD) IL E E E-09 IL-6+VHH6 (2 M) 2.10E E-07 NA VHH6 immobilized kd (1/s) SE (kd) KD (M) IL-6+gp80 (2 M) 3.09E E-07 NA gp80+il-6 (2 M) 3.75E E-07 NA gp130 immobilized ka (1/Ms) kd (1/s) gp80 [IL-6 (2 M)] ^ gp80 [IL-6 (2 M)+VHH6 (2 M)] ^ IL-6 [gp80 (2 M)] ^ IL-6 [gp80 (2 M) + VHH6 (2 M)] ^ SI Table 3: Additional SPR data analysis. During SPR studies binding of IL-6, gp80 and VHH6 were individually tested in a concentration series (0-250 nm, as two-fold serial dilution). When proteins were tested in combinations (IL-6 and gp80, IL-6 and VHH6, gp80 and VHH6, gp80 and IL-6 and VHH6) one of the proteins was titrated in a concentration series (0-250 nm), while the other was kept constant at an excess concentration of 2 µm (in brackets) to ensure complex formation. A total of four proteins were immobilized on the chip, from top to bottom: IL-6, gp80, VHH6 and gp130.
13 13 SI Table 4: In-house constructs Construct pnafl-8his- fusionil- 6(gp80D123) pnafl-gp80d123- hscfc Expression Host Promoter Affinity Tag Expressed Protein CHO CMV 8His gp80(l20- S320)(C211A, C277A)- IL-6(V30-M212) CHO CMV Single chain gp80(m1- human IgG Fc P322)(C211A, C277A)- fragment hscfc pnafl-8his-gp80v4 CHO CMV 8His gp80v4 ptrx-6his-hil-6 E.coli T7 Thioredoxin- 6His Trx-6His-IL-6(A28- M212) ptrx-6his-il-6(s21) E.coli T7 Thioredoxin- 6His Trx-6His-IL-6(S49- M212) pimms-6his-vhh6 Expi-HEK CMV 6His VHH6-6His SI Table 4: In-house designed constructs. All constructs listed in the table were designed in-house to support the immunization campaign, antibody screening, junctional antibody characterization and biophysics analysis.
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